Nucleic acids are set apart from other biomolecules by their ability to hybridize to complementary sequences, a feature that is exploited in nature for the replication of genetic information. Hybridization specificity is exploited in research and diagnostics to generate information about the presence and quantity of nucleic acid sequences. Hybridization assays are generally based on the specific binding of a single stranded analyte to a labeled single stranded probe, followed by detection of the resulting duplexes. Variations of this basic scheme have been developed to enhance specificity, facilitate the separation of the duplexes from extraneous materials, and/or amplify the detectable signal.
The development of solid phase oligonucleotide synthesis has greatly simplified the production of specific nucleic acid probe and primers. Synthetic probes are widely used for all aspects of nucleic acid diagnosis, therapy and investigation. A feature that can be provided only in synthesized probes is comb-type branched multimers, which are composed of a linear backbone and pendant side chains. The backbone includes a segment that provides a specific hybridization site for a nucleic acid of interest, while the pendant side chains include iterations of a segment that provide specific hybridization sites to a second sequence of interest. The branch points are typically provided by protected phosphoramidites, as described in U.S. Pat. No. 5,359,100 (Urdea et al.); U.S. Pat. No. 5,656,731 (Urdea); U.S. Pat. No. 5,124,246 (Urdea et al.) and U.S. Pat. No. 5,710,264 (Urdea et al.), which are introduced during the oligonucleotide synthesis. The branch points may by symmetric or asymmetric.
An appealing aspect of synthetic primers is the ability to tag the probe as it is synthesized, thereby eliminating a separate labeling procedure. Common tags include internal or terminal tags or spacers, where an attached detectable label may be fluorescein or other fluorochromes, or a binding moiety such as biotin, digoxigenin, etc. Spacers known in the art include those with a 2-aminobutyl-1,3-propanediol backbone (U.S. Pat. No. 5,451,463), which is incorporated during phosphoramidite synthesis.
Detection of specific genetic sequences is an area of active research and development. 1X However, many problems still exist, such as low levels of signal, small sample size, high sample complexity, and the like. Improvements in the ability to provide a multiplicity of labels to a specific probe sequence are of interest, particularly using reagents that are compatible with standard phosphoramidite synthesis. The present invention addresses these issues.
The synthesis of multiple-label carriers using DNA synthesis chemistry is disclosed in U.S. Pat. No. 5,359,100 (Urdea); European Patent EP 0 292 128 (Segev), and WO 90/00622 (Kwiatkowski et al.) The use of triethylene glycol as a building block is described in U.S. Pat. No. 4,914,210 (Leveason et al.) The basic method for solid phase DNA synthesis using phosphoramidite chemistry is described in U.S. Pat. No. 4.458,066, issued Jul. 3, 1984; U.S. Pat. No. 4,500,707, issued Feb. 19, 1985; and U.S. Pat. No. 5,153,319, issued Oct. 6, 1992. Reagents and protocols are widely available, for example from Applied Biosystems, Inc. (Foster City, Calif.). Branching phosphoramidites are commercially available, for example from Clontech (Palo Alto, Calif.).
Nucleic acid probes having highly hydrophilic non-nucleosidic tags with multiple labels are provided. The tags are branched structures synthesized using solid phase phosphoramidite chemistry, generally in combination with synthesis of the nucleic acid portion of the probe. The building blocks of the tag are protected glycerol, mono- and di-ethylene glycol phosphoramidites; and reagents that introduce attachment sites for labels. The resulting tag structure permits introduction of multiple labels, while enhancing the hydrophilicity of the probes through additional phosphodiester moieties.
It is to be understood that this invention is not limited to the particular methodology, protocols, constructs, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
As used herein the singular forms xe2x80x9caxe2x80x9d, xe2x80x9candxe2x80x9d, and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to xe2x80x9ca probexe2x80x9d includes a plurality of such probes and reference to xe2x80x9cthe structurexe2x80x9d includes reference to one or more such structures and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.
A probe refers to a biopolymer comprising a nucleic acid moiety and a tag moiety. A labeled probe further comprises one or more detectable label moieties covalently or non-covalently attached to the attachment sites provided by the tag moiety. The nucleic acid sequence is complementary to a nucleic acid sequence of interest present in a target analyte.
As used herein, the term target region or target nucleotide sequence refers to a probe binding region contained within the target molecule. The term target sequence refers to a sequence with which a probe will form a stable hybrid under desired conditions.
The nucleic acid moiety of the probe as used herein is conventional. The length, degeneracy, and specific sequence of the nucleic acid moiety is determined largely by the use for which it is intended. Generally the length of a particular strand will be sufficiently long to provide for specific hybridization of the sequence of interest, and sufficiently short to provide for a difference in hybridization between the sequence of interest and other sequences such as may be present in the sample. For example, detection of a single base change in a genetic sequence may be accomplished with probes of from about 12 to 25 nucleotides in length. Multiple strands may be combined in a comb or fork-like structure.
It will be appreciated that the binding sequences need not have perfect complementarity to provide stable hybrids. In many situations, stable hybrids will form where fewer than about 10% of the bases are mismatches, ignoring loops of four or more nucleotides. Accordingly, as used herein the term xe2x80x9ccomplementaryxe2x80x9d refers to an oligonucleotide that forms a stable duplex with its xe2x80x9ccomplementxe2x80x9d under assay conditions, generally where there is about 90% or greater homology.
The nucleic acid moiety is typically synthesized in vitro using standard chemistry, and may be naturally occurring, e.g. DNA or RNA, or may be synthetic analogs, as known in the art. Such analogs may be preferred for use as probes because of superior stability under assay conditions. Modifications in the native structure, including alterations in the backbone, sugars or heterocyclic bases, have been shown to increase intracellular stability and binding affinity. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoramidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3xe2x80x2xe2x80x94Oxe2x80x2xe2x80x945xe2x80x2xe2x80x94S-phosphorothioate, 3xe2x80x2xe2x88x92Sxe2x80x945xe2x80x2xe2x80x94O-phosphorothioate, 3xe2x80x2xe2x80x94CH2xe2x80x945xe2x80x2xe2x80x94O-phosphonate and 3xe2x80x2xe2x80x94NHxe2x80x945xe2x80x2xe2x80x94O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage.
Sugar modifications are also used to enhance stability and affinity. The a-anomer of deoxyribose may be used, where the base is inverted with respect to the natural b-anomer. The 2xe2x80x2xe2x80x94OH of the ribose sugar may be altered to form 2xe2x80x2xe2x80x94O-methyl or 2xe2x80x2xe2x80x94O-allyl sugars, which provides resistance to degradation without compromising affinity.
Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2xe2x80x2-deoxycytidine and 5-bromo-2xe2x80x2-deoxycytidine for deoxycytidine. 5-Propynyl-2xe2x80x2-deoxyuridine and 5-propynyl-2xe2x80x2-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.
These terms include double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, xe2x80x9ccapsxe2x80x9d, those containing pendant moieties, such as, for example, proteins, including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc., those with intercalators, e.g. acridine, psoralen, etc., those containing chelators, e.g. metals, radioactive metals, boron, oxidative metals, etc., those containing alkylators, those with modified linkages, e.g. alpha anomeric nucleic acids, etc., as well as unmodified forms of the polynucleotide or oligonucleotide.
The term polynucleotide analyte refers to a single- or double-stranded nucleic acid molecule that contains a target nucleotide sequence. The analyte nucleic acids may be from a variety of sources, e.g. biological fluids or solids, food stuffs, environmental materials, etc., and may be prepared for the hybridization analysis by a variety of means, e.g. proteinase K/SDS, chaotropic salts, or the like. The term xe2x80x9cpolynucleotide analytexe2x80x9d is used interchangeably herein with the terms xe2x80x9canalyte,xe2x80x9d xe2x80x9canalyte nucleic acidxe2x80x9d and xe2x80x9ctarget.xe2x80x9d
As used herein, a biological sample refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, semen, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents, e.g. conditioned medium resulting from the growth of cells in cell culture medium, virus-infected cells, recombinant cells, and cell components. Exemplary uses of the subject probes are in detecting and/or quantitating: viral nucleic acids, such as from hepatitis B virus (xe2x80x9cHBVxe2x80x9d), hepatitis C virus (xe2x80x9cHCVxe2x80x9d), hepatitis D virus (xe2x80x9cHDVxe2x80x9d), human immunodeficiency virus (xe2x80x9cHIVxe2x80x9d), and the herpes family of viruses, including herpes zoster (chicken pox), herpes simplex virus I and II, cytomegalovirus, Epstein-Barr virus, Herpes VI virus; bacterial nucleic acids, such as Chlamydia, Mycobacterium, etc.; and numerous human sequences of interest.
The term nonspecific hybridization is used to refer to those occurrences in which a segment of a first polynucleotide which is intended to hybridize to a segment of a selected second polynucleotide also hybridizes to a third polynucleotide, triggering an erroneous result, i.e. giving rise to a situation where label may be detected in the absence of target analyte. The use of the term xe2x80x9chybridizesxe2x80x9d is not meant to exclude non-Watson-Crick base pairing.
Nonspecific binding refers to those occurrences in which a polynucleotide binds to a solid support, or other assay component, through an interaction, which may be either direct or indirect, that does not involve hydrogen bonding to support-bound polynucleotides.
The tag moiety has a basic structure as shown below: 
where Lp is a spacing monomer, Np is an internal monomer providing an attachment site for a label, tNp (terminal Np) is an internal monomer as previously defined br a monovalent 5xe2x80x2 amino-modifer, and Yp is a bi-branching monomer. Independently, n is from about 0 to 20, usually from about 1 to 10, n1 is from about 0 to 20, usually from about 1 to 10, n2 is from about 0 to 20, usually from about 1 to 10, x is from about 0 to 9, and y is from about 0 to 9. One or more of the basic structures may be incorporated into the tag.
For clarity, the reactants used to produce the tag moiety are herein referred to generically as reactant monomers, and specifically as reactant-Np, reactant Lp, reactant-Yp, etc. The reactant monomers will typically have at least one phosphoramidite group, and at least one protected group suitable for chain extension, e.g. DMT or levulinyl protected hydroxyl groups. The reactant monomer may further comprise a functional group for branch elongation (reactant-Yp) or for label attachment (reactant-Np). The reactant-tNp may lack the protected group for chain extension.
Yp is a bi-branching non-nucleosidic monomer that has three functional groups for branching off each elongating chain of polymer. In one embodiment of the invention, Yp has the structure: 
Reactant-Yp comprises protected hydroxyl groups at each the branch points, where the protecting group may be the same or different, in order to provide for symmetric or asymmetric branching, respectively. Reactant Yp has the general structure: 
wherein R1 and R2 are independently chosen from hydrogen and lower alkyl, R3 is xcex2-cyanoethyl or methyl, and R4 is a protecting group for a primary hydroxyl group.
Np is an attachment site monomer that provides a functional group suitable for post-synthetic attachment of a label. The functional group providing an attachment site is compatible with phosphoramidite synthesis. Groups of particular interest are amino groups, e.g. Uni-Link amino modifier, LCA-phosphoramidite, etc. Reactant-Np comprises an attachment site group and a protected group for chain extension. Reactant-tNp comprises only an attachment site group.
Examples of suitable reactant-tNp compounds include: 
It is preferred that both Np and tNP provide an amino group.
Lp is a spacing non-nucleoside monomer. The reactant-Lp comprises two or more functional groups, two of which are needed and utilized for elongating the chain of the polymer. In one embodiment of the invention, Lp has the structure: 
where n is 0 or 1.
Reactant Lp has the general structure: 
wherein R1 and R2 are independently chosen from hydrogen and lower alkyl, R3 is xcex2-cyanoethyl or methyl, and R4 is a protecting group for a primary hydroxyl group.
A label as used herein refers to a detectable moiety, which may be a direct or indirect signal generating compounds. Suitable labels include fluorochromes, e.g. acridinium esters (AE), fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2xe2x80x2,7xe2x80x2-dimethoxy-4xe2x80x2,5xe2x80x2-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2xe2x80x2,4xe2x80x2,7xe2x80x2,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, e.g. 32P, 35S, 3H; light generating enzyme molecules, e.g. alkaline phosphatase on the stable dioxetane systems, etc. The label may be a two stage system, where the amplified DNA is conjugated to a binding compound, e.g. biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner in turn carries or is covalently linked to direct signal generating molecules. The label may also provide a specific cleavage site, e.g., an enzymatic cleavage site.
Labels are attached to the tag moiety through the reactive groups provided by Np monomers. Generally a labeled probe will comprise multiple labels, ranging from at least about 5 label moieties to as much as 100 or more label moieties.
In a first embodiment, the invention provides a probe, comprising a nucleic acid moiety; and at least one tag moiety, wherein said tag moiety has the structure: 
where Lp is a spacing monomer, Np is an internal monomer providing an attachment site for a label, tNp is an internal monomer as previously defined or a monovalent 5xe2x80x2 amino-modifier, and Yp is a bi-branching monomer, independently, n is from 0 to 20, n1 is from 0 to 20, n2 is from about 0to 20, x is from 0 to 9, and y is from 0 to 9.
The tags of the nucleic acid probes are highly hydrophilic non-nucleosidic moieties that include multiple labels. The tags are branched structures having a phosphodiester backbone, which are synthesized using solid phase phosphoramidite chemistry, generally in combination with synthesis of the nucleic acid portion of the probe. The tag structure permits introduction of multiple labels, while enhancing the hydrophilicity of the probes through additional phosphodiester moieties.
The nucleic acid moiety of probes is conventional, utilizing any of a number of oligonucleotide chemistries as known in the art. The specific sequence, length, complexity and degeneracy of the nucleotide sequence may vary according to the specific intended use. Probes are labeled with any of a variety of detectable compounds, particularly fluorochromes and high affinity binding moieties, e.g. avidin, digoxigenin, etc.
An example of a branched non-nucleosidic tag has the structure: 
The monomers of the subject compounds serve at least two functions in the tag, one as a negative charge unit and the other as a spacing unit, in addition to the branching and label attachment functions. In structure 6, Lp* represents a tri-ethylene glycol unit introduced as its DMT protected phosphoramite, Lp represents a mono- or di-ethylene glycol unit introduced as compounds 8 and 9 below, Yp represents a bi-branching monomer introduced as compound 7, and Np represents any reagents to introduce a free amine on to the carrier for label attachment. This branched structure is shown in the orientation from 5xe2x80x2 to 3xe2x80x2. If the branched carrier needs to be at the 3xe2x80x2 end of a DNA sequence, 5xe2x80x2 to 3xe2x80x2 DNA synthesis chemistry should be employed (reagents available from Glen Research, Sterling, Va.; see Science (1988) 241:551-557 for method).
Two examples of 6 were synthesized using Np of Uni-Link AminoModifier (Example 6), and Np of LCA phosphoramidite (Example 8). The subject tag compounds allow an amine group, and therefore a label attachment site, to be introduced at any position in the sequence, in order to facilitate the multiplicity of labels.
The reactant monomers for synthesis of the branched tag 6 have been synthesized, or are commercially available. Reactant monomers typically have at least one phosphoramidite group and at least one protected group hydroxyl for chain elongation. The reactant monomer may further comprise a second protected hydroxyl for branch synthesis, or a functional group for label attachment.
Three reactant monomer compounds have been synthesized, with the following structures. An exemplary reactant Yp has the structure: 
Exemplary reactant monomers for Lp have structures as shown below: 
Synthesis of the non-nucleosidic branched tag uses the same reaction chemistry as phoramidite synthesis of oligonucleotides. A reactive phosphorous group of one monomer is coupled to the 5xe2x80x2 hydroxyl of another monomer. Generally the tag will be synthesized as an extension of a glass coupled nucleic acid moiety. Acetonitrile is used as a washing solvent and the entire synthesis is performed under argon gas.
The hydroxyl group on the initial glass coupled moiety is protected by a dimethoxytrityl group (DMT). The DMT group is removed with a protic acid, such as trichloroacetic, revealing the reactive hydroxyl. As with polynucleotide synthesis, the released DMT ion is strongly colored and can easily be quantified by colorimetry or conductivity. The monomer for coupling is added with tetrazole to protonate the nitrogen of the phosphoramidite, making it susceptible to nucleophilic attack. Typically the reactive monomers will be synthesized as cyanoethyl phosphoramidites,
Unlike oligonucleotide synthesis, a capping step for failure sequences, which remain uncoupled after reaction, is optional, because deletion of a single monomer will not necessarily affect the tag function in a substantial way. If it is performed, capping may be accomplished by acetylation with acetic anhydride and 1-methylimidizole. The coupled phosphite chain is then oxidized, e.g. with iodine as oxidizer and water as the oxygen donor to change the trivalent phosphoramidite to a pentavalent phosphate triester. At the end of synthesis, the finished probe is cleaved from the solid support with a strong base, e.g. ammonia. Following synthesis, the attachment groups are deprotected, if necessary, and used for label attachment with methods as known in the art.
For compound 7, with two bulky flanking DMT protective groups present, the coupling to a free hydroxy group is slow. However, by using double coupling of 10 min. each, the coupling yield reaches 88.5% to 94.5%, leaving its amplification factor of 1.77 to 1.89 (theoretical maximum of 2.0). Similarly with reactant 8, having a short distance between the DMT group and phosphorus, the coupling reaction is also slow. Using double coupling of 10 min each, the coupling yield reaches 96.7 to 100%. For compound 9, the coupling yield is always near quantitative as the regular DNA synthesis phosphoramidite reagents.
It has been previously postulated that 7 undergoes internal cyclization, however, the high coupling yields indicate that the internal cyclization is not a significant factor. In the synthesis of compound 10 below, the average stepwise coupling yield was 98.1% by manual trityl coloremetric measurement, after 30 units of compound 8 (MEG) and 10 units of a long chain amine(LCA) were introduced.
DNA-(MEG3-LCA)10-Txe2x80x83xe2x80x8310
The probes of the subject invention are used in hybridization reactions and assays where a highly labeled probe is desirable. A sample potentially comprising a target nucleic acid is analyzed by one of a number of methods known in the art. Hybridization with probes to Southern blots, dot blots, etc. may be performed. The hybridization pattern of probes to an array of oligonucleotide probes immobilized on a solid support may also be used as a means of detecting the presence of target sequences. For examples of arrays, see Ramsay (1998) Nat. Biotech. 16:40-44; Hacia et al. (1996) Nature Genetics 14:441-447; Lockhart et al. (1996) Nature Biotechnol. 14:1675-1680; and De Risi et al. (1996) Nature Genetics 14:457 -460.
Other applications in which the present invention may find utility include in situ hybridizations, in reducing of nonspecific binding in hybridization assays and in polymerase chain reaction (PCR) assays. In situ hybridization lacks sufficient sensitivity to detect a single molecule of target analyte. In situ PCR (see, for example Bagasra et al. (1993) J. Immunological Methods 158:131-145) has been developed to meet this sensitivity need; however, quantitation is not as precise with the PCR method. An alternative would use the subject probes to bind the target analyte, thereby increasing the signal produced by a single hybridization event. One skilled in the art will recognize that the same strategy could be applied to blot assays, such as dot blots, Southerns, and Northerns, to reduce nonspecific hybridization and nonspecific binding of the probes to the solid supports.
Solution phase capture hybridization may take advantage of the subject probes (see for example, U.S. Pat. No. 5,681,702). Generally the assays proceed as follows. Single-stranded analyte nucleic acid is incubated under hybridization conditions with the appropriate labeled probe. The resulting product is a nucleic acid complex of the analyte polynucleotide bound to the probe. This complex may be subsequently added under hybridizing conditions to a solid phase having capture probes bound to the surface thereof; however, in a preferred embodiment, the initial incubation is carried out in the presence of the support-bound capture probes. The resulting product comprises the complex bound to the solid phase. The solid phase with bound complex is then separated from unbound materials.
In another embodiment of the invention, a cleavage assay is performed. To further reduce the non-specific binding of a tracer or signal probe complex comprising a tag with multiple labels, a gene probe assay employing release-transfer-capture steps is used. A specifically cleavable moiety is included in the construction of the capture moiety, which is then immobilized on a solid surface. After the hybridization reaction with the probe is complete, using a conventional sandwich or branched-DNA assay format, a cleavage step is introduced to release the whole complex from the solid phase, which can then be transferred in solution form to another container to be recaptured as pure desired signal. The non- specifically bound label that is left behind includes various non-specific bound probes, which stick to the surfaces of the solid phase or container wall without specific sequence hybridization. Examples of cleavable moieties include sites for enzymes that recognize nucleic acids specifically or non-specifically, e.g. restriction endonucleases, DNAses, etc., photocleavable moieties, periodate cleavage, etc.
The reactant monomers described herein are used in the synthesis of branched or linear tags, for use in producing highly labeled nucleic acid probes. The advantages are a small dimensional size and high hydrophilicity. After the tag is labeled, its high negative charge and minimal size help to keep the carriers away from DNA or RNA molecules, due to repulsion between negative charges. Non-specific intercalation and steric hindrance are therefore minimized, and the hydrophobicity, if any of reporter molecules is reduced.